Effects And Mechanism Of HS100A2 On Human Colon Cancer

Backgrounds and objectivesColon cancer is the frequent malignant tumor of digestive tract . The 40-50 years old group has the highest incidence of colon cancer, and its incidence in male is 2 to 3 times that of female. The incidence of colon cancer is in front row in Europe and America, it incidence and mortality is merely lower than those of gastric cancer, esophageal cancer and lung cancer in china. The survical rate in 5 years is merely 50% after radical excision. The recurrence and metastasize after operation is the important cause of death. And the incidence is rising year by year. So, it is very important to diagnose and treat it early.S100 protein family is a kind of calcium-binding protein with EF-hands. These proteins play a series of physiological functions, including cell proliferation, transduction of extracellular signal, adhesion among cells, cell mobility, calcium homeostasis, protein,phosphorylation,enzyme activity and transcription factor regulation and so on. At least 25 kinds of protein have been identified as S100 proteins,of which 21 kinds locate in chromosome 1q21. The chromosome segment is instable and rearrangeable that implicates in oncogenesis . S100A2 belongs to the family and its gene locates in chromosome 1q21 as well. High level of S100A2 in tumors means that may be involved in infiltration and metastasis of tumor and prognosis of patients.Cyclooxygenase is also named as prostaglandin peroxides synzyme,it is the criticality rate-limiting enzyme of arachidonic acid metabolism. In the effect of stimulating factor, COX-2 expression obviously up-regulates.It involves in many physiological and pathological process and concerns with effluence, development and metastasis of tumor.Wnt /β-catenin signaling pathway is an important pathway in normal development and pathological process andβ-catenin is a pivotal protein in this pathway.Its high expression can exist in many tumors.This study investigated the effects and possible mechanisms of human S100A2 on colon cancer cells HCT116 and SW480, including in vitro and in vivo. It can provide experimental evidence for interpreting the effect of human S100A2 on tumor development.Methods1. Preparation and identification of human S100A2 recombinant protein:pGST-moluc and pGST-moluc-hS100A2 were transformed into E Coli.(BL21) by CaCl2 method; the expression of GST or GST-S100A2 protein were induced by IPTG; the bacteria lysate were prepare by ultrasound on ice; GST and GST-S100A2 were isolated and purifyed from the lysate with the Glutathione Sepharose 4B beads; the recombinant proteins were identified and quantified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western-blot and Bradford, respectively. Stored in aliquots at -80℃.2. HCT116 and SW480 cells were treated with different concentrations (3, 10, 30, 100 and 300μg/ml) of GST-hS100A2, the concentration dependent of cell proliferation was detected with MTT method.3. Based on the test results, an appropriate GST-hS100A2 concentration was selected to treat HCT116 and SW480 cells. MTT assay was used to measure the survive rate of HCT116 and SW480 treated with GST-hS100A2. And the cell apoptosis was detected by Hoechst33258 dying method; the cell cycle was detected by flow cytometry (PI stain); the cell migration ability was determined by migration assay; the cell invasion ability was determined by the invasion transwell chambers invasion Assay.4. Adenovirus of human S100A2 was amplified and identifited, HCT116 cells were infected with appropriate efficiency of infection and collected to inoculate on nude mouse, observed the effect of human S100A2 on tumorigenesis.5. RT-PCR, Western blot, immunocytochemical method and immunohistochemical method were used to detect the effect of human S100A2 on the levels of COX-2 andβ-catenin. Results1. Target fragment from digestion of pGST-moluc-hS100A2 by HindⅢand EcoRⅠwas about 300bp, which was consistent with restriction enzyme map of pGST-moluc-hS100A2 .The purity of recombinant protein reached 92% and specific positive reaction bands of anti-S100A2 in 36KD appeared in Western blot analysis. 15.7mg protein was obtained from 1L solution which detected by bicinchoninic acid (BCA) protein assay.2.In the extent of detective concentration(3μg/ml to 300μg/ml), exogenous human S100A2 inhibited the proliferation of HCT116 and SW480 cells in a dose-dependent manner. Based on the results, we chose 100μg/ml to treat all cells. GST-human S100A2 (GST-hS100A2) inhibited the proliferation of HCT116 and SW480 cells in a time-dependent manner (P< 0.05).3. Hoechst33258 dying displayed that the apoptosis rates of the HCT116 and SW480 cells in GST-human S100A2 groups were 3.7 and 4.2 times as that of control groups at 48 hours(P<0.05). It indicated that human S100A2 promote HCT116 and SW480 cell apoptosis.4. Flow cytometry displayed that the precent of the G0/G1 phase HCT116 cells in GST-hS100A2 group increased by 49.3% and cells of S phase decreased by 41.6% compared with that of control group at 12 hours(P<0.05). It showed that human S100A2 block cell cycle in G0/G1 phase for HCT116 cell line. 5. The assay of cell migration ability displayed that the HCT116 cell healing rate of 12 hours and 24 hours decreased by 40.7% and 19.2% compared with that of control groups (P<0.05). It showed that human S100A2 inhibit HCT116 cell migration.6. The invasion transwell chambers invasion assay displayed that the HCT116 and SW480 cell numbers of trans-membrane was decreased by 73%and 65%at 24 hours after treated with GST -human S100A2 fusion protein(P<0.05). It prompted that human S100A2 could inhibit cell invasion.7. The results of the assay in vivo showed that the tumor was formation on subcutaeous of the nude mouse after 6 days inoculation with HCT116 cells treated with AdhS100A2, the tumor formation rate of all three groups were the same by 80%. The tumours in AdhS100A2 group growed slowly and their volume was 10.4% of that in control group at the 26 day (P<0.05).There was no any migration in all the three group in vivo.8. RT-PCR results displayed that exogenous human S100A2 decreased gene transcription of COX-2 by 78.4 % (P<0.05) in HCT116 cells. Western blot displayed that exogenous human S100A2 decreased the protein level of COX-2 andβ-catenin in HCT116 cells: the gray ratio of COX-2 in hS100A2 group is 70% of that in control group(P<0.05), and the gray ratio ofβ-catenin in hS100A2 group is 25% of that in control group(P<0.05) ; immunocytochemistry method also displayed that exogenous human S100A2 decreased the protein level of COX-2 andβ-catenin in HCT116 cells. Immunohistochemical method displayed that exogenous human S100A2 decreased the protein level of COX-2 andβ-catenin in subcutaeous tumours, and the average optical density of COX-2 in AdhS100A2 group is 78.7%(P<0.05) of that in control group and the average optical density ofβ-catenin in AdhS100A2 group is 34.9%(P<0.05) of that in control group.Conclusions1. GST-human S100A2 with biologic activity and high purity was prepared by induction expression in E.coli.BL212. Exogenous human S100A2 can inhibit cell proliferation of human colorectal cancer cell lines HCT cells and SW480 cells, promot their apoptosis, block HCT116 cell cycle, inhibit migration and invasion .It can also inhibit the growth of xenografts of HCT116 in nude mice.3. Exogenous human S100A2 can decrease the protein level of COX-2 andβ-catenin. It prompted that the inhibitive effect of human S100A2 on human colorectal cancer cell line might be mediated by inhibiting cox-2 and Wnt /β-catenin signaling pathway.